Cell transplantation into immune compromised mice has transformed our understanding of human disease and has been used extensively to assess regeneration, stem cell self-renewal, and cancer in the xenograft transplantation setting. Despite their great utility, mouse models are not amenable to large-scale studies due to high husbandry costs and do not easily facilitate direct visualization of engrafted cells at single cell resolution. By contrast, zebrafish are inexpensive, can be reared in large numbers, and are amenable to large-scale chemical genetic approaches where compounds can be added directly to the water. Moreover, optically-clear immune-deficient zebrafish strains have permitted large-scale cell transplantation studies to dynamically image fluorescent-labeled cells at single cell resolution. Despite these successes, more needs to be done to develop immune compromised zebrafish as a robust and long-term xenograft cell transplantation model. The long-term goal of this application is to develop a universal zebrafish transplantation model for engrafting a wide array of regenerative and cancer cell types from zebrafish, mouse, and human. The overall objective of this application is to provide new immune deficient zebrafish models for optimized allograft engraftment of regenerative tissues and xenograft engraftment of human cancer, ES, iPS, and CD34+ cord blood cells. The rationale for our research is that zebrafish blood development is highly conserved and that developing zebrafish transplantation models has already led to unique understanding of regenerative stem cell processes and dynamic visualization of new cell behaviors that drive cell growth. Aim 1 will develop compound mutant and humanized transgenic zebrafish for optimized cell transplantation. We will develop new models that lack all T, B, and NK cells, including mutants in the recently identified NK-lysin expressing cytotoxic blood cells and full loss-of-function mutations in the rag2 gene, which is required for mature T and B cell function. We will also generate humanized zebrafish that transgenically express factors that support elevated growth of human cells, including the human ?don?t eat me? signal inhibitory regulatory protein alpha (SIRPa) and human cytokines. Aim 2 will utilize these models for assessing orthotopic and xenograft engraftment, identifying lines that have superior, long-term engraftment of human cancer cell lines, ES and iPS cells, and CD34+ cord blood cells. Aim 3 will refine a system for global distribution and rapid dissemination of mutant lines to the zebrafish, stem cell, and regenerative medicine community. Our work is significant because it will develop a much-needed resource for the community, facilitating the next generation of low-cost, high throughput cell transplantation models to engraft a wide array of regenerative cell types. This work is expected to have a positive translational impact by developing pre-clinical animal models that facilitate direct visualization of engrafted cells at reduced cost and allow chemical genetic approaches to uncover pathways associated with regeneration and stem cell function. Such broad reaching applications for immune compromised zebrafish spans the mission of many NIH institutes.